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Recombinant hog cholera virus for expressing firefly luciferase gene and application of recombinant hog cholera virus

A technology of luciferase gene and swine fever virus, which is applied in the direction of virus/phage, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of high-throughput screening limitations and achieve the effect of saving time

Inactive Publication Date: 2014-09-03
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the detection of green fluorescent protein needs to be judged by fluorescence microscopy, so it is limited in high-throughput screening

Method used

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  • Recombinant hog cholera virus for expressing firefly luciferase gene and application of recombinant hog cholera virus
  • Recombinant hog cholera virus for expressing firefly luciferase gene and application of recombinant hog cholera virus
  • Recombinant hog cholera virus for expressing firefly luciferase gene and application of recombinant hog cholera virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Rescue of recombinant CSFV carrying Fluc gene

[0056] 1. Experimental method

[0057] 1.1 Construction of a full-length infectious clone carrying the Fluc gene

[0058] Primer N was designed according to the Fluc gene in the pGEM-luc plasmid pro Fluc-2-F / N pro Fluc-2-R (Table 1), using this plasmid as a template to amplify the Fluc gene; design primer N according to the full-length sequence of the parent virus Shimen strain pro Fluc-1-F / N pro Fluc-1-R and N pro Fluc-3-F / N pro Fluc-3-R (Table 1), use these two pairs of primers to amplify the gene fragment between the 215th-412th base and the 413th-684th base of the virus respectively; pro Fluc-1-F / N pro Fluc-3-R is a primer, and the fusion fragment Fluc-N is obtained by overlapping PCR pro , and this fusion fragment Fluc-N pro Linked to the pMD18-TSimple vector for sequencing identification.

[0059] Table 1 Primer names and their sequences

[0060]

[0061] Will sequence the correct Fluc-N pro ...

Embodiment 2

[0068] Example 2 Screening of recombinant swine fever virus

[0069] 1. Test method

[0070] 1.1 Luciferase activity analysis of recombinant virus

[0071] The luciferase activity of the five strains of recombinant CSFV rescued in Example 1 was detected with a dual-luciferase detection kit. SK6 cells were inoculated in a 24-well cell culture plate. When the cells grew to 80%, the SK6 cells were infected with the 5 strains of recombinant virus rescued in Example 1 and the Shimen strain of the parental virus by MOI=0.1 dose, and placed in 5% CO 2 , Continue culturing in a 37°C environment. At 12h, 24h, 36h, 48h, 60h, and 72h after infection, the cell culture supernatant was discarded, and the cells were washed once with PBS, and then 100 μL of lysate was added. After lysing on ice for 15 min, the cell lysate was added to the In the white plate of the enzyme detection reagent, immediately put it into the fluorescence detector for reading.

[0072] 1.2 Genetic stability analy...

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Abstract

The invention discloses a recombinant hog cholera virus for expressing firefly luciferase gene and application of the recombinant hog cholera virus, and belongs to the field of rescuing and applying recombinant hog cholera virus for expressing firefly luciferase gene. According to the recombinant hot cholera virus, a firefly luciferase gene is cloned into a Npro protein coding region of a hog cholera virus Shimen strain full infectious cloned pBRCISM by an overlapping polymerase chain reaction method, the recombinant virus CSFV-NproFluc capable of stably expressing firefly luciferase gene is rescued by utilizing an RNA polymerase II reverse genetic operation technology, wherein the preservation number of the recombinant hog cholera virus is CGMCC (China General Microbiological Culture Collection Center) No.9058. The firefly luciferase gene carried by the recombinant hog cholera virus can be stably inherited in the continuous passage process, the hog cholera virus infection does not change. The recombinant hog cholera virus has a wide application prospect in antibody titre determination and high-throughput screening hog cholera virus specificity inhibitors and the like.

Description

technical field [0001] The present invention relates to recombinant swine fever virus, in particular to a strain of recombinant swine fever virus expressing firefly luciferase gene, and the invention also relates to the application of the recombinant swine fever virus in the determination of antibody titer and screening of specific inhibitors of swine fever virus , belongs to the field of rescue and application of recombinant classical swine fever virus expressing firefly luciferase gene. Background technique [0002] Classical swine fever (CSF) is a contact and fatal infectious disease caused by classical swine fever virus (CSFV), characterized by high fever and hemorrhage, and extremely high morbidity and mortality , is listed as an animal disease that must be declared by the World Organization for Animal Health (OIE). Swine fever is a serious hazard to the pig industry and often causes huge economic losses. It is also one of the major animal diseases that China plans to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/85C12Q1/70G01N33/569
Inventor 仇华吉申梁李永锋孙元李素罗玉子
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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